Download Sample Genus Chapter: Methylocella

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gbm00797
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Genus Methylocella
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Defining publication: Dedysh, Liesack, Khmelenina, Suzina, Trotsenko, Semrau, Bares,
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Panikov, and Tiedje 2000, 967VP, emend. Dedysh, Berestovskaya, Vasylieva, Belova,
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Khmelenina, Suzina, Trotsenko, Liesack, and Zavarzin 2004, 154
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Authors: Svetlana N. Dedysh1, Peter F. Dunfield2
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Academy of Sciences, Leninsky Ave. 33/2, Moscow 119071, Russia
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AB, T2N 1N4.
Winogradsky Institute of Microbiology, Research Center of Biotechnology of the Russian
Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary,
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Etymology: Me.thyl.o.cel'la. M.L. n. Methylocella methyl-using cell.
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Abstract:
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Gram-negative, aerobic, polymorphic, slightly curved rods with rounded ends or ovoids. Produce
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large, highly refractile, intracellular poly-ß-hydroxybutyrate granules, one at each pole.
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Reproduce by normal cell division. Cells occur singly or in shapeless aggregates, but do not form
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rosettes. Non-motile. Encapsulated. Cells lack an extensive intracytoplasmic membrane system
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typical of most described methanotrophic bacteria, but contain a vesicular membrane system
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composed of singular flattened or ovoid vesicles connected to the cytoplasmic membrane.
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Facultatively methanotrophic. Methane is oxidized by a soluble methane monooxygenase;
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particulate form of this enzyme is absent. C1 compounds are assimilated via the serine pathway.
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Tricarboxylic acid cycle is complete. Capable of growth on ethanol, some organic acids, ethane,
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propane and some other multicarbon compounds, but sugars are not utilized. Acetate is preferred
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over methane, and leads to a down regulation of methane oxidation. Fix atmospheric nitrogen via
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an oxygen-sensitive nitrogenase. Moderately acidophilic, mesophilic and psychrotolerant. Prefer
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dilute media of low salt content. The major fatty acid is 18:1ω7c. The major quinone is Q-10.
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Members of the class Alphaproteobacteria, family Beijerinckiaceae. Known habitats are acidic
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peatlands and soils.
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Keywords: aerobe, methanotroph, methylotroph, acidic peatlands, soil, serine pathway
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Description:
Gram-negative, aerobic, polymorphic, slightly curved rods with rounded ends or ovoids.
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Produce large, highly refractile, intracellular poly-ß-hydroxybutyrate granules, one at each
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pole. Reproduce by normal cell division. Cells occur singly or in shapeless aggregates, but do
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not form rosettes. Non-motile. Encapsulated. Cells lack an extensive intracytoplasmic
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membrane system typical of most described methanotrophic bacteria, but contain a vesicular
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membrane system composed of singular flattened or ovoid vesicles connected to the
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cytoplasmic membrane. Facultatively methanotrophic. Methane is oxidized by a soluble
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methane monooxygenase; particulate form of this enzyme is absent. C1 compounds are
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assimilated via the serine pathway. Tricarboxylic acid cycle is complete. Capable of growth on
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ethanol, some organic acids, ethane, propane and some other multicarbon compounds, but
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sugars are not utilized. Acetate is preferred over methane, and leads to a downregulation of
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methane oxidation. Fix atmospheric nitrogen via an oxygen-sensitive nitrogenase. Moderately
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acidophilic, mesophilic and psychrotolerant. Prefer dilute media of low salt content. The major
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fatty acid is 18:1ω7c. The major quinone is Q-10. Members of the class Alphaproteobacteria,
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family Beijerinckiaceae. Known habitats are acidic peatlands and soils.
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DNA G + C content (mol %): 61.2 – 63.3 (Tm).
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Type species: Methylocella palustris Dedysh, Liesack, Khmelenina, Suzina, Trotsenko, Semrau,
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Bares, Panikov, and Tiedje 2000, 967VP.
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Number of species with validated names: 3.
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Family classification:
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Beijerinckiaceae (fbm00164)
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Further Descriptive Information
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Cell morphology and ultrastructure. Three currently described species of the genus
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Methylocella, i.e. M. palustris, M. silvestris and M. tundrae, were isolated from acidic peat bogs,
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acidic forest soil and acidic tundra wetland, respectively (Dedysh et al., 2000; Dunfield et al,
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2003; Dedysh et al., 2004). Cells of all species are Gram-negative, non-motile, polymorphic
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rods. M. palustris and M. silvestris form slightly curved rods with rounded ends, 0.6-1.0 µm
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wide and 1.0-2.5 µm long (Fig. 1a), while cells of M. tundrae are only 1.0-1.5 µm long, and form
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short rods or ovoids (Table 1). Old cultures of M. tundrae contain a large number of cells that
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under phase-contrast microscopy appear phase-light in the middle and phase-dark on both edges.
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Cells of Methylocella species reproduce by normal cell division and occur singly or in shapeless
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aggregates, but do not form rosettes. The major distinctive feature of the cells is their bipolar
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appearance, which is easily observable under phase-contrast microscopy (Fig. 1a). This
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appearance is due to large, highly refractile, intracellular granules of poly-ß-hydroxybuturate,
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which form at each cell pole (Fig. 1b). When grown on solid media, cells of M. palustris and M.
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silvestris produce large polysaccharide capsules up to 1 µm thick, which can be stained with
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ruthenium red. Each cell, therefore, is separated from the others by the capsular material. In
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contrast to M. palustris and M. silvestris, cells of M. tundrae do not produce a macrocapsule.
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Although the rare appearance of exospore-like cell forms in several-month-old cultures was
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reported in the original description of M. palustris (Dedysh et al., 2000), this has not been
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confirmed by further studies. Cysts or cyst-like cells are also not formed by these bacteria.
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The cell ultrastructure in Methylocella species is unusual compared to most characterized
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proteobacterial methanotrophs. The extensive, stacked intracytoplasmic membrane structures
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found in most other methanotrophs are absent from Methylocella cells. Instead, the cells contain
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a vesicular membrane system composed of small (40–100 nm in diameter) spherical, ovoid, or
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tube-shaped vesicles formed by cytoplasmic membrane invaginations (Fig. 1c). These vesicles
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are bounded by three-layered membranes, and each contains a homogenous matrix of lower
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electron density than the cytoplasm. To date this unique cell ultrastructure has been described
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only for Methylocella species and for the closely related methanotroph Methyloferula stellata
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(Vorobev et al., 2011).
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Colonial and cultural characteristics. On agar media, visible colonies of Methylocella species
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appear after 2-3 weeks of incubation. After growth on plates for 6 weeks, the colonies of M.
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palustris are highly raised, have a tough slimy consistency, are circular with an entire margin and
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a smooth surface, and are 1-2 mm in diameter. Colonies 2-3 months-old may have a folded
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brain-like surface texture. Initially, the colonies are semi-transparent or uniformly turbid, but
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become opaque white over time. Six-week-old colonies of M. silvestris are also raised and
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circular but slightly larger in size, up to 2–4 mm in diameter. With continued growth, the closely
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located colonies (1-5 mm distance) may merge together to form an amorphous slimy cover on
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the agar surface. Colonies of M. tundrae are less raised and not slimy. They are circular,
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opaque/cream-colored and 1–3 mm in diameter. Liquid cultures of Methylocella species display
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white turbidity; a surface pellicle is not formed. Flocks of biomass are commonly formed in
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liquid cultures of M. palustris and M. silvestris, while M. tundrae shows homogenous growth
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(Table 1).
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Nutrition and growth conditions. Like all other methanotrophs, members of the genus
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Methylocella are able to grow on methane as the sole carbon and energy source. However, the
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key enzyme of most currently known methanotrophic bacteria, i.e. particulate methane
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monooxygenase (pMMO), is lacking in Methylocella species. Instead, these methanotrophs
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possess only a soluble methane monooxygenase (sMMO). Among the known aerobic
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methanotrophs, the absence of pMMO-encoding genes and the presence of only an operon
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encoding sMMO (mmoXYBZDC) are features shared only by Methylocella species and
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Methyloferula stellata (Chen et al., 2010; Dedysh et al., 2015; see gbm01403). Comparative
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sequence analysis of MmoX (the alpha subunit of the hydroxylase component of sMMO) shows
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that a distinct lineage of this gene groups Methylocella spp. and Methyloferula stellata apart
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from type I methanotrophs and the Methylocystis/Methylosinus (see fbm00169) group of type II
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methanotrophs (Figure 2).
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Originally, Methylocella species were described as aerobic bacteria capable of growth on
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only the C1 compounds methane, methanol, methylamine and formate. Later, however, it was
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shown that they also grow on acetate, pyruvate, succinate, malate and ethanol (Dedysh et al.,
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2005), while sugars are not utilized. Acetate is preferred over methane. The growth rate and
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carbon conversion efficiency are higher on acetate than on methane, and when both substrates
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are provided in excess acetate is preferentially used and methane oxidation is shut down.
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Methanol is utilized in a wide range of concentrations, but the three different species possess
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different optima (Table 1). The range of growth substrates was further extended for M. silvestris,
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which was demonstrated to utilize propane, ethane, propanol, propanediol, acetone, methyl
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acetate, acetol, glycerol, propionate, tetrahydrofuran, and gluconate (Crombie, Murrell, 2014).
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Whether or not these substrates are are also suitable for M. palustris and M.tundrae has not been
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verified yet. Since the description of Methylocella silvestris as the first facultative methanotroph,
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a few other methanotroph species have also been shown to consume either acetate or ethanol
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(Dedysh, Dunfield, 2011; Dunfield, Dedysh, 2014). However, the diversity of substrates utilized
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by Methylocella silvestris by far exceeds that of any other known methanotrophic bacterium
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(Dunfield, Dedysh, 2014).
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All members of the genus Methylocella utilize ammonium salts, nitrates, yeast extract
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and some amino acids as nitrogen sources. They use the glutamate cycle for NH4+ assimilation.
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When grown in nitrogen-free medium, they are able to fix N2 via an oxygen-sensitive
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nitrogenase.
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The temperature range for growth of Methylocella spp. is 4–30°C. Different species
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possess slightly different optima (Table 1). They grow between pH 4.2 and 7.5, with an optimum
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at pH 5.0–6.0. They are highly sensitive to salt stress and, therefore, prefer diluted media with a
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low salt content (0.2–0.5 g L-1). Thus, members of the genus Methylocella can be characterized
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as psychrotolerant mesophiles and moderate acidophiles with a low salt tolerance.
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Chemotaxonomic characteristics. Similarly to other methanotrophic and methylotrophic
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representatives of the family Beijerinckiaceae, the major fatty acid in Methylocella species is 11-
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cis-octadecenoic acid (18:1ω7c). It comprises 78-82% of the total fatty acids in M. palustris and
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M. silvestris, and 59-62% in M. tundrae. Besides 18:1ω7c, cells of M. tundrae also contain
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significant amounts (7-13%) of 16:1ω7c and 19:0 ω8c cyclo fatty acids. Notably, 10-
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cisoctadecenoic acid (18:1 ω8c), which is highly characteristic of alphaproteobacterial
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methanotrophs belonging to the family Methylocystaceae (see fbm00169), is not present in cells
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of Methylocella species.
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Genome features. Among the three described species of Methylocella, a genome sequence has
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to date been determined only for M. silvestris BL2T (Chen et al., 2010). The genome size is 4.3
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Mb. Two identical rRNA operons and 3917 predicted protein-coding genes were identified. The
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closure of the complete circular genome has conclusively verified the absence of any pmoCAB
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genes encoding pMMO. Instead, a complete operon encoding sMMO (mmoXYBZDC) is present.
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In addition to the mmo operon, a gene cluster encoding an additional soluble di-iron center
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monooxygenase (SDIMO) was detected in the genome of M. silvestris BL2T. This second
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SDIMO was shown to be a propane monooxygenase, which acts together with sMMO to
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facilitate propane oxidation by this unusual methanotroph (Crombie, Murrell, 2014).
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A complete operon encoding methanol dehydrogenase (mxaFJGIRSACKLDEH) and all
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genes necessary for fixation of methane-derived carbon via the serine cycle are present in the
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genome. However unlike in many other alphaproteobacterial methylotrophs the ethylmalonyl
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CoA pathway for regenerating glyoxylate for the serine cycle is missing (Tamas et al., 2013).
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Instead genes encoding glyoxylate bypass enzymes (i.e., isocitrate lyase and malate synthase) are
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present and probably assist in the assimilation of carbon for both 1-C and 2-C substrates
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(Crombie, Murrell, 2011). The ability to grow on the C2 compound acetate as well as on some
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C3 and C4 compounds is explained by the presence of a full gene set encoding enzymes of the
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tricarboxylic acid (TCA) cycle, including genes encoding α-ketoglutarate dehydrogenase, which
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are lacking in some other methanotrophs. Acetate kinase- and phosphotransacetylase-encoding
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genes are also present, allowing acetate to be fed into the TCA cycle.
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Ecology. Members of the three described species of the genus Methylocella were isolated from
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various mildly acidic environments, i.e. Sphagnum-dominated peat bogs, temperate forest soil,
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and tundra wetland (Dedysh et al., 2000, 2004; Dunfield et al., 2003). Further use of cultivation-
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independent 16S rRNA gene-based methods demonstrated that Methylocella species are widely
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distributed in acidic and neutral terrestrial environments. Methylocella-like 16S rRNA gene
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sequences have been retrieved from neutral (pH 6.8) and acidic (pH 4.2-4.8) peatlands (Morris et
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al., 2002; Chen et al., 2008), acidic (pH 3.5-4.0) forest soils (Radajewski et al., 2002; Lau et al.,
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2007), and a neutral (pH 6.2) landfill cover soil (Chen et al., 2007). Using fluorescence in situ
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hybridization with species-specific, 16S rRNA-targeted oligonucleotide probes, Methylocella
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palustris was enumerated at greater than 106 cells per g of wet peat in a Sphagnum peat bog
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(Dedysh et al., 2001).
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Due to the absence of pMMO in Methylocella species, these bacteria cannot be detected
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using a pmoA-based PCR assay considered universal and specific for all other known
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methanotrophs. To overcome this limitation, a Methylocella-specific mmoX-targeted assay was
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developed to detect and enumerate these methanotrophs in environmental samples (Rahman et
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al., 2011). It was revealed that Methylocella species are not restricted to acidic environments and
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can also be detected in neutral and alkaline ecosystems, although their population size is
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generally higher in acidic habitats. The abundance of Methylocella in selected samples of
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sediments, soils and peats determined with this assay was in the range 0.9-3.3 × 106 cells g-1.
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Enrichment and Isolation Procedures
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A key to successful isolation of Methylocella spp. to date has been the use of moderately acidic
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(pH 5.0-5.8) mineral media with a low salt content. One suitable medium for these
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methanotrophs is liquid mineral medium M2 of the following composition (g per liter
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demineralized water): KNO3, 0.25; KH2PO4, 0.1; MgSO4 × 7H2O, 0.05; CaCl2 × 2H2O, 0.01;
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NaCl, 0.02; 0.1% (v/v) of trace element solution; pH 5.0-5.5. Trace element solution has the
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following composition (g per liter distilled water): Na2EDTA, 0.5; FeSO4 × 7H2O, 0.2; H3BO3,
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0.03; ZnSO4 × 7H2O, 0.01; MnCl2 × 4H2O, 0.003; CoCl2 × 6H2O, 0.02; CuSO4 × 5H2O, 0.03;
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NiCl2 × 6H2O, 0.002, Na2MoO4 × 2H2O, 0.003. Alternatively, diluted nitrate mineral agar salts
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(DNMS) medium at pH 5.8 as described by Dunfield et al. (2003) can also be used. In the case
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of methane-rich environments, isolation efforts can start by adding serial dilutions of
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environmental samples directly to solid media on Petri plates and incubating these under a
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methane-enriched atmosphere. A more reliable approach, however, involves obtaining
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enrichment cultures of methanotrophs. The sample of interest is used to inoculate a respective
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liquid mineral medium and is incubated under a headspace enriched in methane (10-30%, v/v) in
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static conditions for 3-6 weeks. Isolation of methanotrophs from the resulting enrichment
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cultures is achieved by plating an aliquot of the respective cell suspensions on agar medium M2
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or DNMS. Solid media can also be prepared with gellan gum (Gel-Gro; ICN Biomedicals).
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Inoculated plates are incubated for 1-1.5 months at 20-24 °C in a closed glass desiccator
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containing a headspace of 20% (v/v) methane and 5% CO2 (v/v) in air. The colonies appearing
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on the plates are randomly picked for examination by phase microscopy for the presence of
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slightly curved thick rods with bipolar appearance. This procedure is continued until individual
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colonies of target bacteria are ultimately identified and obtained in pure culture. Additional tips
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for isolation and purification of methanotrophic bacteria are described by Dedysh & Dunfield
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(2014).
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Maintenance Procedures
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Strains can be maintained either on the solid media M2 and DNMS media or in liquid cultures.
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For growth in liquid media, screw-capped serum bottles are used with a headspace/liquid space
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ratio of 4 : 1. After inoculation, methanol (0.25-0.5%, v/v) is added aseptically to the cultures
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and the bottles are capped with silicone rubber septa to prevent loss of methanol by evaporation,
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or methane is added aseptically through silicone rubber septa to achieve a mixing ratio in the gas
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headspace of approximately 10–20 %. Bottles are incubated on a rotary shaker (100-120 r.p.m.)
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at 20-24 °C. Strains are sub-cultured once every 1.5-2 months. Although acetate is the preferred
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growth substrate of Methylocella species, media with acetate are not recommended for
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maintaining these methanotrophs due to the risk of contamination by heterotrophic bacteria.
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Long term storage can be achieved by addition of 5-10% dimethyl sulfoxide and freezing at -80
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C. We have retrieved samples preserved for >10 years under these conditions.
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Procedures for testing special characteristics
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Procedures for verifying methanotrophic activity and distinguishing between obligate and
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facultative methanotrophy are described in detail by Dedysh & Dunfield (2011). The presence of
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sMMO can be confirmed using the colorimetric naphthalene oxidation test (Graham et al., 1992)
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and by means of PCR-mediated amplification of mmoX gene encoding the α-subunit of the
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sMMO hydroxylase. The mmoX gene fragment can be amplified from DNA of Methylocella
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species using a combination of the primers mmoXA-166f (5’- ACCAAGGARCARTTCAAG-3’)
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and mmoXD-1402r (5’- TGGCACTCRTARCGCTC-3’) (Auman et al., 2000), which are
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applicable for most sMMO-containing methanotrophs. Alternatively, Methylocella-specific
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mmoX-targeted primers mmoXLF (5’-GAAGATTGGGGCGGCATCTG-3’) and mmoXLR (5’-
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CCCAATCATCGCTGAAGGAGT-3’) developed by Rahman et al. (2011) can be used. One
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additional effective tool for detecting Methylocella cells is fluorescence in situ hybridization
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with 16S rRNA-targeted oligonucleotide probes. Several species-specific probes have been
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developed for members of this genus including Mcell-1026 for M. palustris (5’-
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GTTCTCGCCACCCGAAGT-3’), Mcells-1024 for M. silvestris (5’-
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TCCGGCCAGCCTAACTGA-3’) and Mcellt-143 for M. tundrae (5’-
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TTCCCCGAGTTGTTCCGA-3’) (Dedysh et al., 2001; 2005).
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Differentiation of the genus Methylocella from other genera
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The ability to grow on methane clearly differentiates Methylocella from non-methanotrophic
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members of the family Beijerinckiaceae, i.e. the genera Beijerinckia (see gbm00795),
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Methylovirgula (see gbm01405) and Methylorosula (see gbm01404). Characteristics that
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distinguish Methylocella from other methanotrophs of the family Beijerinckiaceae are listed in
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Table 2. Cell morphology, the absence of intracytoplasmic membranes, absence of pMMO, and
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the preference for growth on acetate makes Methylocella different from members of the genus
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Methylocapsa (see gbm01402). The inability to form rosettes and to grow at pH below 4, the
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absence of RubisCO activity, and the ability to utilize multicarbon compounds distinguishes
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Methylocella from Methyloferula (see gbm01403) species.
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Taxonomic comments
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The closest neighbors of Methylocella based on 16S rRNA gene sequence phylogeny are
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sMMO-possessing obligate methanotrophs of the genus Methyloferula (see gbm01403),
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heterotrophs of the genus Beijerinckia, pMMO-possessing methanotrophs of the genus
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Methylocapsa (see gbm01402) and facultative methylotrophs of the genera Methylovirgula (see
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gbm01405) and Methylorosula (see gbm01404). All these metabolically distinct bacteria display
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96-97% 16S rRNA gene sequence similarity to each other (Figure 3). Taxonomic construction
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based on 21 concatenated methylotrophy genes verifies the same close phylogenetic relationship
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of Methylocella to Methylocapsa and Beijerinckia (Tamas et al., 2013).
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269 List of species of the genus Methylocella
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1. Methylocella palustris Dedysh, Liesack, Khmelenina, Suzina, Trotsenko, Semrau, Bares,
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Panikov, and Tiedje 2000, 967VP.
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pa.lus´tris. M.L. adj. palustris bog-inhabiting
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Description as for the genus plus the following traits. Cells are polymorphic, slightly curved
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bipolar rods, 0.6-1.0 µm wide by 1.0-2.5 µm long. Produce large polysaccharide capsules.
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Colonies of are highly raised, semi-transparent or uniformly turbid, have a tough slime
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consistency, are circular with an entire margin and a smooth surface. Optimal growth at 20°C
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and at pH 5.5. Carbon sources used include methane, methanol, acetate, ethanol, pyruvate,
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succinate, malate. Methanol is utilized in low concentrations, up to 0.3% (v/v). Nitrogen sources
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are ammonium salts, nitrates and yeast extract. NaCl inhibits growth at a concentration of 0.5%
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(w/v). The type strain was isolated from Sphagnum peat from the Kyrgyznoye ombrotrophic bog
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in West Siberia (56° N, 85° E). The species also includes strains S6 and M131.
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DNA G + C content (mol %): 61.2 (Tm).
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Type strain: K, ATCC 700799.
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EMBL/GenBank accession (16S rRNA gene): Y17144.
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2. Methylocella silvestris Dunfield, Khmelenina, Suzina, Trotsenko, and Dedysh 2003, 1238VP.
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sil.ves´tris. L. adj. silvestris of the forest
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Cells are slightly curved bipolar rods, 0.6–0.8 µm in width and 1.2–1.5 µm in length. Produce
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large polysaccharide capsules. Colonies are raised and circular, semi-transparent or uniformly
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turbid. Optimal growth occurs at 15–25°C and at pH 5.5. Capable of slow growth at 4°C. Carbon
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sources used include methane, methanol, methylamines, acetate, ethanol, pyruvate, succinate,
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malate, propanol, propanediol, acetone, methyl acetate, acetol, glycerol, propionate,
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tetrahydrofuran, gluconate, ethane, propane. Methanol is utilized in a wide concentration range,
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from 0.01 to 5% (v/v). NaCl inhibits growth at concentrations above 0.8% (w/v). The type strain
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was isolated from an acidic cambisol under a beech-dominated forest near Marburg, Germany.
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The species also includes strain A1.
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DNA G + C content (mol %): 63.0% (genome analysis).
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Type strain: BL2, DSM 15510, NCIMB 13906.
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EMBL/GenBank accession (16S rRNA gene): AJ491847.
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EMBL/GenBank accession (genome): CP001280.
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3. Methylocella tundrae Dedysh, Berestovskaya, Vasylieva, Belova, Khmelenina, Suzina,
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Trotsenko, Liesack, and Zavarzin 2004, 155VP.
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tun´drae. N.L. gen. fem. n. tundra from the tundra, the northern zone of Eurasia and North
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America
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Cells grown on methane are curved ovoids. Old cultures contain many cells that appear phase
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light in the middle and phase dark on both edges. Cells do not possess a macrocapsule and
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colonies are not slimy like those of M. palustris or M. silvestris. Liquid cultures display
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homogeneous turbidity. Optimal growth occurs at 15°C and at pH 5.5–6.0. Capable of slow
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growth at 5°C and pH 4.2. Carbon sources include methane, methanol, methylamine, formate,
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acetate, ethanol, pyruvate, succinate, malate. Methanol is utilized in a wide range of
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concentrations from 0.01 to 2.0% (v/v). NaCl inhibits growth at concentrations above 0.8%
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(w/v). The distinctive feature of the PLFA profile is the presence of 19:0ω8c cyclo fatty acids.
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The type strain was isolated from an acidic Sphagnum peatland in Vorkuta region, northern
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Russia. The species also includes strains TCh1 and TY1.
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DNA G + C content (mol %): 63.3 (Tm).
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Type strain: T4, DSM 15673, NCIMB 13949.
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EMBL/GenBank accession (16S rRNA gene): AJ555244.
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Other species
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“Methylocella imadethisoneupus” Whitman and Dedysh, 2016, 155.
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i mad e thi son’e up us N.L. gen. masc. n. imadethisoneupus a hypothetical name to show how to
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include species whose names have not been validated in your genus chapter.
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The “other species” section can be used for species whose names have not been validated,
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species whose names have been validated but then transferred to other genera, and species that
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have been misassigned to a genus. If the species name is not validated, the names is in quotes
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and includes the effective publication. Notice the absence of the superscripts “VP” or “AP”. For
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species that have been transferred or misassigned, include a brief rationale for not including the
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species in the “List of species”. A more extensive discussion can be given in the genus
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description under the section “Taxonomic Comments”. Otherwise, the description should have
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similar content as used for the “List of Species”.
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DNA G + C content (mol %): 00.0 (Lc).
335
Type strain: None, DSM 0, NCIMB 0.
336
EMBL/GenBank accession (16S rRNA gene): Z0000R0.
337
338
339
REFERENCES
340
Auman AJ, Stolyar S, Costello AM, & Lidstrom ME (2000) Molecular characterization of
341
methanotrophic isolates from freshwater lake sediment. Appl Environ Microbiol 66: 5259-
342
5266.
343
Chen Y, Dumont MG, Cebron A, & Murrell JC (2007) Identification of active methanotrophs in
344
a landfill cover soil through detection of expression of 16S rRNA and functional genes.
345
Environ Microbiol 9: 2855-2869.
346
Chen Y, Dumont MG, McNamara NP, Chamberlain PM, Bodrossy L, Stralis-Paverse N, &
347
Murrell JC (2008) Diversity of the active methanotrophic community in acidic peatlands as
348
assessed by mRNA and SIP-PLFA analyses. Environ Microbiol 10: 446-459.
349
Chen Y, Crombie A, Tanvir Rahman M, Dedysh S, Liesack W, Stott MB, Alam M, Theisen AR,
350
Murrell JC, & Dunfield PF (2010) Complete genome sequence of the aerobic facultative
351
methanotroph Methylocella silvestris BL2. J Bacteriol 192: 3840-3841.
352
Crombie A & Murrell JC (2011) Genetic systems for the facultative methanotroph Methylocella
353
silvestris. In: Methods in Enzymology, vol. 495, Rosenzweig and Ragsdale (eds) Academic
354
press (Elsevier Inc.), Burlington; pp 119-133.
355
356
357
Crombie AT & Murrell JC (2014) Trace-gas metabolic versatility of the facultative
methanotroph Methylocella silvestris. Nature 510: 148-151.
Dedysh SN, Liesack W, Khmelenina VN, Suzina NE, Trotsenko YA, Semrau JD, Bares AM,
358
Panikov NS & Tiedje JM (2000) Methylocella palustris gen. nov., sp. nov., a new methane-
359
oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-
360
pathway methanotrophs. Int J Syst Evol Microbiol 50: 955-969.
361
Dedysh SN, Derakshani M, & Liesack W (2001) Detection and enumeration of methanotrophs in
362
acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridization, including the use of
363
newly developed oligonucleotide probes for Methylocella palustris. Appl Environ Microbiol
364
67: 4850-4857.
365
Dedysh SN, Berestovskaja YY, Vasylieva LV, Belova SE, Khmelenina VN, Suzina NE, Trotsenko
366
YA, Liesack W & Zavarzin GA (2004) Methylocella tundrae sp. nov., a novel methanotrophic
367
bacterium from acidic tundra peatlands. Int J Syst Evol Microbiol 54: 151-156.
368
Dedysh SN & Dunfield PF (2011) Facultative and obligate methanotrophs: how to identify and
369
differentiate them. In: Methods in Enzymology, vol. 495: Rosenzweig and Ragsdale (eds).
370
Academic press (Elsevier Inc.), Burlington; p 31-44.
371
Dedysh SN & Dunfield PF (2014) Cultivation of methanotrophs. In: Hydrocarbon and Lipid
372
Microbiology Protocols, McGenity et al. (eds). Springer Protocols Handbooks, Springer,
373
Berlin, doi 10.1007/8623_2014_14.
374
375
376
Dunfield PF & Dedysh SN (2014) Methylocella: a gourmand among methanotrophs. Trends
Microbiol 22: 368-369.
Dunfield PF, Khmelenina VN, Suzina NE, Trotsenko YA & Dedysh SN (2003) Methylocella
377
silvestris sp. nov. a novel methanotrophic bacterium isolated from an acidic forest cambisol.
378
Int J Syst Evol Microbiol 53: 1231-1239.
379
Graham DW, Korich DG, LeBlanc RP, Sinclair NA & Arnold RG (1992) Applications of a
380
colorimetric plate assay for soluble methane monooxygenase activity. Appl Environ Microbiol
381
58: 2231–2236.
382
Lau E, Ahmad A, Steudler PA, & Cavanaugh CM (2007) Molecular characterization of
383
methanotroph communities in forest soils that consume atmospheric methane. FEMS
384
Microbiol Ecol 60: 490-500.
385
Morris SA, Radajewski S, Willison TW, & Murrell JC (2002) Identification of the functionally
386
active methanotroph population in a peat soil microcosm by stable-isotope probing. Appl
387
Environ Microbiol 68:1446-1453.
388
Radajewski S, Webster G, Reay DS, Morris SA, Ineson P, Nedwell DB, Prosser JI, & Murrell JC
389
(2002) Identification of active methylotroph populations in an acidic forest soil by stable
390
isotope probing. Microbiology 148:2331-2342.
391
Rahman MT, Crombie A, Chen Y, Stralis-Pavese N, Bodrossy L, Meir P, McNamara NP, &
392
Murrell JC (2011) Environmental distribution and abundance of the facultative methanotroph
393
Methylocella. ISME J 5: 1061–1066.
394
395
Tamas I, Smirnova AV, He Z, & Dunfield PF (2014) The (d)evolution of methanotrophy in the
Beijerinckiaceae, a comparative genomics analysis. ISME J 8: 369-382.
396
Vorobev AV, Baani M, Doronina NV, Brady AL, Liesack W, Dunfield PF, & Dedysh SN (2011)
397
Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately methanotrophic
398
bacterium possessing only a soluble methane monooxygenase. Int J Syst Evol Microbiol 61:
399
2456-2463.
400
401
Table 1. Characteristics that differentiate described species of the genus Methylocella.
Characteristic
M. palustris
M. silvestris
Cell morphology
Polymorphic, slightly
curved bipolar rods
Slightly curved bipolar Short, slightly
rods
curved rods or
ovoids
Cell size, μм
0.6-1.0 × 1.0-2.5
0.6-0.8 × 1.2-2.0
0.6-0.8 × 1.0-1.5
Macrocapsule
formation
+
+
-
Highly raised, tough
slime, circular and semitransparent
Growth in liquid
media
Slimy and aggregated
Raised, white or semitransparent, circular
colonies with an entire
edge and a smooth
surface
Slimy and aggregated
Optimal growth
temperature, ˚С
20
20-25
15
Optimal growth pH
5.0 - 5.5
5.5
5.5-6.0
Growth on CH3OH,
% (v/v)
≤ 0.3
≤ 5.0
≤ 2.0
18:1ω7c
18:1ω7c
18:1ω7c, 16:1ω7c,
19:0 ω8c cyclo
61.2
63.0*
63.3
Colony morphology
Major fatty acids
DNA G+C content,
Tm
402
M. tundrae
*- data based on genome sequence analysis
Less raised, not
slimy, circular,
opaque/creamcolored
Homogenous
403
Table 2. Major characteristics that distinguish Methylocella from other methanotrophic members
404
of the family Beijerinckiaceae
Characteristic
Cell morphology
Cell size, μm
Bipolar straight or
curved rods
Straight or curved rods
0.6 – 1.0 × 1.0 – 2.5
0.4 – 0.65 × 1.1 – 3.0
Methylocapsa
Curved coccoids
0.7 – 1.2 × 0.8 – 3.1
+
–
Facultative
methanotrophy
Obligate
methanotrophy
Obligate or limited
facultative
methanotrophy
+
+
+
-
Methanol, acetate
Methanol
Methane
acetate, ethanol,
pyruvate, succinate,
malate, propanol,
propanediol, acetone,
methyl acetate, acetol,
glycerol, propionate,
tetrahydrofuran,
gluconate, ethane,
propane
None
None or acetate
RubisCO activity
_
+
_
Growth at/in:
pH 3.5
0.5% NaCl
–
–
+
+
–
–
60-63.3
59.5
61.4-61.9
Type of metabolism
Possession of:
pMMO
sMMO
Preferred growth
substrate(s)
Multicarbon
compounds utilized
G+C content (mol%)
406
Methyloferula
–
Rosette formation
405
Methylocella
407
FIGURE CAPTIONS
408
Figure 1. (a) Phase-contrast micrograph of cells of Methylocella silvestris BL2T; bar, 10 µm. (b,
409
c) Electron micrographs of ultrathin sections of vegetative cells of Methylocella palustris KT;
410
bars, 0.5 µm. PHB, granules of poly-ß-hydroxybutyrate; PP, granules of polyphosphate; MV,
411
membrane vesicles.
412
413
Figure 2. Unrooted neighbor-joining tree constructed based on 368 deduced amino acid sites of
414
partial mmoX gene sequences, showing the positions of Methylocella species relative to other
415
sMMO-possessing type I and type II methanotrophs. Bootstrap values (1000 data resamplings)
416
>80% are shown. Bar, 0.05 substitutions per amino acid position.
417
418
Figure 3. 16S rRNA gene-based maximum-likelihood tree showing the phylogenetic position of
419
Methylocella species in relation to other representatives of the Beijerinckiaceae. The type I
420
methanotrophs Methylomicrobium album (X72777), Methylobacter luteus (AF304195),
421
Methylomonas methanica S1 (AF304196) and Methylococcus capsulatus Texas (NR_029241)
422
were used as an outgroup (not shown). Bar, 0.05 substitutions per nucleotide position.
423
424
425
426
427
428
429
430
431
432
433
434
435
436
Figure 1.
437
438
439
Figure 2
440
441
442
443
444
445
446
447
448
449
450
Figure 3.